We continued an investigation of the structure of mammanlian DNA polymerase Alpha. Our current results confirm that a 190 KDa polypeptide is an Alpha-polymerase catalytic subunit in growth phase monkey BSC-1 cells and that additional catalytic subunits of about 115 KDa and 70 KDa are present also. The 190 KDa polypeptide can be obtained directly from crude soluble extracts of growing cells by immunoprecipitation with antibody to Alpha-polymerase and is enzymatically active after electroelution from an SDS-polyacrylamide gel. Further improvements in our use of immunoblotting techniques have enabled detection of alpha-polymerase polypeptides in both crude extracts of mammalian cells and extracts of E. coli infected with an expression vector (Lambdagtll) containing mammalian cDNA inserts. Five phage capable of expressing Alpha-polymerase polypeptides have been cloned. A similar approach has been used to obtain other phage clones capable of expressing Beta-polymerase polypeptide and heliz destabilizing protein-1 polypeptide, respectively. Finally, experiments have been conducted toward developing a system for study of polyoma virus DNA replication in vitro. We have obtained evidence that de novo initiation and semiconservative replication occur in reaction mixtures containing plasmid DNA and extract from polyoma virus infected cells. Known requirements of this replication system include 1) the presence in the plasmid DNA of the polyoma virus origin of replication and 2) that the extract comes from infected rather than uninfedcted cells.